Development of an MFG-Based Retroviral Vector System for Secretion

doi:10.1006/mthe.2001.0423, available online at http://www.idealibrary.com on IDEAL

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Development of an MFG-Based Retroviral Vector System for Secretion of High Levels of Functionally Active Human BMP4 Hairong Peng,1,* Shin-Tai Chen,1 Jon E. Wergedal,1 John M. Polo,2 Jiing-Kuan Yee,3 K.-H. William Lau,1†, and David J. Baylink1 1

Department of Medicine, Loma Linda University, and Musculoskeletal Disease Center (151), Jerry L. Pettis VA Medical Center, Loma Linda, California 92357, USA 2 Chiron Corporation, Emeryville, California 94608, USA 3 City of Hope, Duarte, California 91010, USA *Current address: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA

†

To whom correspondence and reprint requests should be addressed. Fax: (909) 796-1680. E-mail: [email protected].

We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 ␮g/106 cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein. Key words: retroviral vector (MFG), hybrid vector, bone morphogenetic protein-4, bone formation, marrow stromal cells

INTRODUCTION Bone morphogenetic proteins (BMPs) have a role in stimulating differentiation of osteoblasts and chondrocytes [1–3]. Accordingly, BMP2, BMP4, and BMP7 induce endochondral bone formation and repair bone defects in various animal models [4–9]. Thus, BMPs have therapeutic potential in bone repair and regeneration. Past studies in various animal models with BMP-protein-based therapy to increase bone formation resulted in success in some instances but not in others [7,10,11]. The lack of success is not due to the BMP protein, because such therapy has been shown to be consistently effective in vitro. It seems probable that the problem lies more in the ability of the carrier/vehicle to achieve physiologic delivery kinetics for an adequate length of time. This can be accomplished, but it may be difficult to achieve with current technology. Gene-based therapy may overcome this limitation.

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Accordingly, the production of BMP by stromal cells is a more physiological means of delivery than the dissolution of BMP protein carriers. In addition, the duration of action may be more appropriate for bone formation with gene therapy. Consequently, gene therapy using various viral vectors expressing a BMP gene to treat skeletal defects has received a great deal of interest. Most past studies on gene therapy with BMPs used adenoviral vectors. Although the results of previous studies using adenoviral vectors expressing BMP2 or BMP7 have been impressive [12–19], adenoviral vectors have a disadvantage in that these vectors, even with the use of “gutted” adenoviral vectors, frequently elicited substantial immune responses. The immune response could produce adverse effects in the animal and, as such, would not allow for sustained expression of the therapeutic gene and would

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FIG. 1. Replacement of BMP4 propeptide with BMP2 propeptide increased secretion of mature BMP4 by transduced cells. Top, the molecular structure of the BMP2/4HA and BMP4–HA constructs. The shaded boxes represent sequences derived from BMP2. Bottom, the western blot comparing the levels of BMP4–HA fusion protein expressed from 293T cells transfected with the retroviral vector plasmid containing BMP2/4–HA hybrid (CBMP2/4HA, simplified from pLNCBMP2/4HA) or that containing the parental construct (CBMP4HA). We analyzed both CM and cell lysate. Unless stated otherwise, we loaded 20 ␮l of each sample in each lane. The apparent molecular mass of mature rhBMP4 monomer is approximately 18 kDa. The 20 kDa band represents the mature BMP4–HA monomer, whereas the 52-kDa band represents monomer of proBMP4HA. LNCX, sample from control empty vector. We repeated this experiment three times and obtained identical results in each repeat experiment.

prevent repeated viral administration. In this regard, retroviral vectors do not elicit substantial immune responses and, therefore, may have an important advantage over adenoviral vectors. In addition, to appropriately treat large bony defects or to prevent or treat chronic bone-wasting disorders, such as osteopenia and osteoporosis, a large and relatively long-term increase in bone formation through a sustained expression of a bone formation gene would be preferred. Accordingly, retroviral vectors allow stable gene integration, whereas adenoviral vectors would not. Consequently, retroviral vectors may have an additional advantage over adenoviral vectors. Research on retroviral vectors expressing BMPs has been limited, because most of the available retroviral vectors do not induce sufficiently high production of BMPs even in highly transfected immortalized cell lines [20], and it has been difficult to generate stable producer clones that produce high-titer retroviruses expressing the BMP gene [21]. Although progress has been made using weaker promoters to drive the expression of the BMP gene [21] and generating viral vectors by means of transient transfection [20], the ability to increase the level of secretion of BMPs in retroviral-transfected cells remains a substantial challenge. We sought to develop a retroviral vector system that could lead to high secretion of BMP4 in transduced

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normal cells that could be used in ex vivo gene therapy to stimulate bone formation. We chose BMP4 for this study for several reasons: (1) BMP4 was expressed in bone and surrounding soft tissues [22]; (2) upon femoral fracture, BMP4 expression was transiently increased tenfold, whereas the expression level of BMP2, BMP5, BMP6, and BMP7 did not change [22]; and (3) during distraction osteogenesis mechanical tension-stress induces expression of BMP4 and BMP2, but not that of BMP6 or BMP7 [23]. Here we describe the development of an MFG-based retroviral vector system that led to high expression and secretion of BMP4 in transduced normal rat marrow stromal cells (as well as skin fibroblasts). The transduced cells, when implanted subcutaneously in syngenic immune competent rats, induced ectopic bone formation, indicating that the secreted BMP4 was functional and that this retroviral vector system could be used in gene therapy to induce bone formation.

RESULTS To facilitate the monitoring and quantitation of BMP4 expression with commercially available anti-hemaglutinin (HA) tag antibodies, we initially developed retroviral vectors expressing human BMP4 fused with a HA tag. We compared the effectiveness of the BMP4–HA expression cassette in three retroviral vectors using different viral promoters to drive transgene expression: LXSN (with MLVLTR promoter), LNSX (with SV40 early promoter), and LNCX (with CMV promoter) [24]. LBMP4HA (BMP4–HA construct in LXSN vector) was consistently the most effective vector of the three for stable viral vector-mediated expression of BMP4, but CBMP4HA (BMP4–HA construct in LNCX vector) was most suitable for transient plasmidmediated expression (data not shown). Thus, we carried out transient plasmid-mediated expression studies to optimize the BMP4 expression construct only with LNCXbased vectors. Conversely, we used vectors with the LTR promoter in studies to produce clones that stably generated BMP4-expressing retrovirus. At any event, the secretion level of mature BMP4–HA protein, even in LBMP4HAtransduced 293T cells, was very low (62 ng/106 cells/24 h conditioned medium (CM)). The replacement of the propeptide of BMP4 with that of BMP2 has been shown to increase the expression of BMP4 protein with a plasmid-based vector [25]. Accordingly, we next tested whether replacement of the BMP4 propeptide domain with the counterpart domain of BMP2 would improve the secretion of mature BMP4 in our vector system. The level of BMP4–HA secreted into CM by 293T cells transfected with the pLNCBMP4HA plasmid (without the BMP2 propeptide replacement) was very low (that is, barely detectable; Fig. 1). Conversely, the pLNCBMP2/4HA hybrid plasmid (with the BMP2 propeptide replacement) produced a considerably higher level of mature BMP4–HA secretion into CM compared with the BMP4–HA-transfected cells.

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FIG. 2. Addition of the HA tag had no effects on BMP4 synthesis and secretion in transduced cells. We compared retroviral vector plasmids containing BMP2/4 hybrid with or without the HA tag (top BMP2/4 versus BMP2/4–HA) for BMP4 expression in CM and cell lysate of 293T cells 24 h after transfection. Unless stated otherwise, we loaded 20 ␮l of each sample. LNCX, sample from control empty vector. We repeated this experiment three times and obtained identical results in each repeat experiment.

The BMP4–HA proprotein (that is, 52 kDa protein) level in cell lysate of the pLNCBMP2/4HA transfected cells was not significantly different from cells transfected with the CBMP4HA vector. We used a plasmid with a reporter gene (GFP) in the same LNCX backbone to assess transfection efficiency, which indicated a 10% variation in transfection efficiency (data not shown). Thus, the difference in the secretion of BMP4–HA protein between the two plasmids was probably not due to differences in transfection efficiency. To assess whether the HA tag would affect the biosynthesis and/or secretion of BMP4 protein, we compared the amount of BMP4 released in the CM and in cell lysate of 293T cells transfected with the retroviral vector plasmid containing the BMP2/4 hybrid construct either with (pLNCBMP2/4HA) or without (pLNCBMP2/4) the HA tag. The level of BMP4 secreted into the CM or that of the proprotein in cell lysate did not differ significantly (Fig. 2), whether or not the HA tag was added to the carboxy terminus of the BMP4 cDNA, suggesting that the HA tag did not alter the biosynthesis and/or secretion of mature BMP4 protein in 293T cells. To determine the effect of the HA tag on BMP4 biological activity, we compared the biological activity of BMP4 produced by cells transfected with either the BMP4 or the BMP2/4 hybrid constructs, with or without the HA tag. The biological activity of BMP4 was assessed by its ability to induce the expression of alkaline phosphatase (ALP) in C2C12 cells. A significant number of cells expressed BMP4 immunoreactive activity in cells transfected with the pLNCBM2/4, pLNCBMP2/4HA, or pLNCBMP4HA vector plasmids (Fig. 3A). When we measured BMP4 protein in the CM of the transfected cells, we only detected a significant amount of BMP protein in the CM of cells transfected with

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vector plasmids containing the BMP2 propeptide (pLNCBMP2/4 and pLNCBMP2/4HA), and not in the CM from cells transfected with vector plasmids without the BMP2 propeptide (pLNCBMP4 and pLNCBMP4HA; pLNCBMP2/4 versus pLNCBMP4, P ⬍ 0.001; and pLNCBMP2/4HA versus pLNCBMP4HA, P ⬍ 0.005; Fig. 3C). However, when we assessed the biological activity of the secreted BMP4, we found that only the CM of the pLNCBMP2/4-transfected cells, not that of cells transfected with the pLNCBMP4HA or pLNCBMP2/4HA vector plasmid, induced the expression of ALP activity in C2C12 cells after a 24-hour treatment (Fig. 3B). Similarly, only cells transfected with the pLNCBMP2/4 vector plasmid, not the pLNCBMP2/4HA vector plasmid, increased the specific activity of cellular ALP in C2C12 cells (pLNCBMP2/4 versus each test vector plasmid, P ⬍ 0.01 for each; Fig. 3D). Thus, these findings indicate that the addition of the HA tag to the C terminus of BMP4 completely abolishes its biological activity. To test whether the enhancement in BMP4 secretion requires the entire propeptide of BMP2, we prepared a BMP2/4 expression construct with a truncated BMP2 propeptide domain (that is, bp 110–774 were deleted). Cells transfected with the parental vector plasmid (pLNCBMP2/4) produced substantial levels of BMP4, but the amount of both the pro- and mature forms of BMP4 in 293T cells transfected with the truncated construct (pLNCBMP2/4SM1 and pLNCBMP2/4SM2) was below the detection level (Fig. 4). The CM of cells transfected with the truncated vector also did not contain detectable levels of mature BMP4 (data not shown). Thus, an intact BMP2 propeptide domain may be essential for BMP4 biosynthesis and secretion. We next subcloned the BMP2/4 hybrid construct into an MFG-based retroviral vector (which was also called splicing retroviral vector [26]). We termed the resulting vector pCLSABMP2/4 (Fig. 5A). We also prepared a reporter vector (pCLSA␤-gal) with the identical backbone but containing lacZ as a control. We then generated retrovirus pseudotyped with the VSV-G envelope containing the BMP2/4 (and LacZ) vector and used these retroviruses to transduce human fibrosarcoma cell line HT1080 at the multiplicity of infection of approximately 20. A high level (1–3 ␮g/106 cells/24 h) of BMP4 was secreted into the CM of the transduced cells two weeks after the transduction (Fig. 5B). The secreted BMP4 was biologically active in the in vitro BMP4 biological assay, and its relative specific activity was comparable to that of recombinant human BMP4 (rhBMP4; data not shown). We then used the VSV-G pseudotyped pCLSABMP2/4 retrovirus to transduce normal rat marrow stromal cells, and we measured the amount of mature BMP4 protein in the CM one and two weeks later, after three rounds of transduction. The average transduction efficiency of this retrovirus in normal rat marrow stromal cells (estimated by determining the percentage of the cells that expressed immunoreactive BMP4 two days after transduction) was

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A

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FIG. 3. Addition of the HA tag to the C terminus inactivated the biological activity of BMP4 protein. (A) Immunocytochemical staining for BMP4 in cells transfected with each indicated BMP4 expression retroviral vector plasmid. (B) Cytochemical staining for alkaline phosphatase (ALP) in C2C12 cells that were treated for 24 h with the CM from cells transfected with retroviral vector plasmids containing BMP4 constructs with or without the HA tag. (C) BMP4 protein levels in the CM of cells transfected with retroviral vector plasmid containing BMP4 constructs with or without the HA tag (CBMP2/4 and CBMP2/4HA, respectively). The results are mean ± S.D. of three independent western blots (CBMP2/4 versus CBMP2/4HA, P ⫽ N.S.; CBMP2/4 versus CBMP4, P ⬍ 0.001; CBMP2/4HA versus CBMP4HA, P ⬍ 0.005). (D) BMP4 biological activity (that is, increased ALP expression in the CM of transfected cells). We derived data of the BMP4 biological activity assay from the same CM used in the BMP-4 western blot analyses (C). Results are mean ± S.D. of three experiments (CBMP2/4 versus all other treatments, P ⬍0.01 each). We determined statistical differences by two-tailed Student’s t test.

50 ± 7.9% (mean ± S.D.). The concentration of mature BMP4 protein in the CM at 1 week was below the detection limit (⬍ 10 ng/106 cells/24 h), but at 2 weeks, the amount was between 37 and 62 ng/106 cells/24 hours. However, the lysate of cells remaining in culture wells contained significant amounts of mature BMP4 at each time point: 25–125 ng/106 cells at 1 week, and 250–375 ng/106 cells at 2 weeks post-transduction. BMPs contain heparin-binding sites and may bind extracellular matrix [27]. Thus, it is possible that some of the secreted BMPs might be trapped in the extracellular matrix [28]. To determine if the BMP4 in the cell lysate

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represented intracellular or secreted but matrix-trapped BMP4, we compared the amounts of BMP4 in cell lysate that was prepared in two ways. First, we lysed cells directly in culture wells. This cell lysate should contain both intracellular BMP4 and secreted BMP4 trapped in the extracellular matrix. Second, we lysed the isolated cells that were released from culture wells with a brief trypsin treatment. The isolated cell lysate should contain only intracellular BMP4. Mature BMP4 was detected only in cell lysate prepared without the trypsinization (Fig. 6), suggesting that the BMP4 detected in lysate of the transfected rat marrow stromal cells (RMSC) might represent secreted BMP4 that

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FIG. 4. Intact propeptide domain is required for BMP4 synthesis and secretion in transfected 293T cells. We compared the level of cellular BMP4 in cells transfected with truncated vectors, CBMP2/4SM 1 and 2 (with deletion in BMP2 propeptide domain as indicated by the area between the dashed line in the top panel), with that in cells transfected with the parent construct CBMP2/4.

was bound to extracellular matrices. Inasmuch as transient transfection is an efficient way to produce retrovirus stock, it would be advantageous to have a stable virus producer cell clone that could potentially provide a consistent and constant supply of high-quality viruses, which might have important benefits in gene therapy. Accordingly, we next sought to produce optimal retrovirus producer clones in an amphotropic packaging cell line, HA-LB [29]. Following transduction with VSV-G pseudotyped pCLSABMP2/4 retroviruses, we subjected HALB cells to single-cell cloning by the limited dilution approach. We screened individual clones (grown out from a single cell) for production of virus stock that caused a high secretion of BMP4 in transduced HT1080 cells. We selected 86 clones and further tested 12 of them for their ability to transduce normal rat marrow stromal cells to secrete mature BMP4. We eventually chose three producer clones for further testing. Virus stocks produced from each of these three clones transduced efficiently (with an average transduction rate of 50 ± 7.9%) and caused secretion of high concentrations of mature BMP4 from transfected normal marrow stromal cells, normal rat dermal fibroblasts, and HT1080 cells (Fig. 7). Initially, up to 1 ␮g/106 cells of BMP4 was detected in the CM and cell lysate of transduced marrow stromal cells (or dermal fibroblasts). However, the amount of BMP4 produced and secreted by the transduced cells decreased progressively with culturing time, reducing to a concentration of 110–180 ng/106 cells/24 hours in CM and 500 ng/106 cells in cell lysate 5 weeks after transduction (data not shown). To determine the in vivo bone induction activity of

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FIG. 5. High production of BMP4 in HT1080 cells transduced with an MFGbased BMP2/4 retroviral vector. (A) Schematic structure of the retroviral vector containing BMP2/4 hybrid (pCLSABMP2/4) or the ␤-gal reporter gene (pCLSA␤-gal). This is an MFG-based vector that contains an intron located between the splice donor (SD) and splice acceptor (SA). ␺, The packaging sequence; CMV-P, the CMV early promoter. (B) BMP4 western blot of the CM and cell lysate of HT1080 cells two weeks after retroviral transduction. PB1 and PB2 represent two independent transductions with the VSV-G pseudotyped BMP4 expression vectors pCLSABMP2/4. ␤-Gal indicates cells transduced by pCLSA␤-gal.

pCLSABMP2/4 retroviral vector, we implanted gelfoam disks impregnated with normal rat marrow stromal cells, which were transduced with either a selected retrovirus clone containing the pCLSABMP2/4 expression construct or retroviruses containing the pCLSA␤-gal construct (expressing ␤gal), subcutaneously in syngeneic rats. Ectopic bone formation (detected by the Faxitron X-ray analysis) was evident in animals receiving cells transduced with the pCLSABMP2/4 retrovirus, starting from two weeks postimplantation (data not shown). In contrast, animals implanted with cells transduced with the pCLSA␤-gal virus showed no evidence of ectopic bone formation. At four weeks postimplantation, all four animals treated with the transduced cells producing BMP-4 showed evidence for ectopic bone formation through visual inspection and Faxitron X-ray analyses of isolated implants. Conversely, none of the 4 control animals receiving the ␤-gal expressing cells showed any ectopic bone formation, even after 12 weeks postimplantation. Histological analysis of sections of the implant site of the animals receiving BMP4 producing transduced marrow stromal cells at four weeks post-transplantation (Figs. 8A–8C) indicated that mineralized bone nodules were formed within the implant site, as demonstrated by the strong Von Kossa

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FIG. 6. Evidence for extracellular matrix sequestration of secreted BMP4 in rat marrow stromal cells (RMSC) transduced with the VSV-G pseudotyped MFGbased BMP2/4 retrovirus. We detected mature BMP4 protein only in cell lysates prepared without trypsinization, not those with a brief trypsin treatment, indicating an extracellular localization of BMP4. PB1 and PB2 represent samples from two independent transductions by BMP4 expression vectors pCLSABMP2/4. ␤-gal represents samples from cells transduced by pCLSA␤-gal.

staining (Fig. 8A). Toluidine blue staining of the sections demonstrated the presence of plump osteoblasts on the surface of the nodules, consistent with active bone formation (Fig. 8B). Consistent with the conclusion that they were of osteoblast lineage, the cells on the surface of the nodules stained strongly for ALP, a known marker of osteoblasts (Fig. 8C). At 12 weeks post-transplanation, well-organized marrow space was seen within some of the bone nodules (Fig. 8D). By contrast, no histomorphometric evidence for ectopic bone formation was seen at the implant site of any of the control animals receiving rat marrow stromal cells transduced with the ␤-gal expressing retrovirus ex vivo (Fig. 8E).

DISCUSSION

tively high-titer retrovirus expressing BMP4. We showed that the ex vivo transduced normal rat marrow stromal cells with our clonal retrovirus induced ectopic bone formation when implanted subcutaneously in immune competent syngeneic rats. These findings support our premise that this MFG-based retroviral vector system will be useful in the retroviral-based BMP4 gene therapy of bone. Three characteristics of this MFG-based BMP4 retroviral vector system are noteworthy. First, this system used an optimized viral promoter to drive expression of the BMP4 transgene in transduced cells. Second, this system used a BMP2/4 hybrid expression construct, in which the propeptide domain of BMP4 was swapped with the corresponding propeptide domain of BMP2, to construct the BMP4 expression retroviral vector. The rationale for the use of this BMP2/4 hybrid construct instead of the native BMP4 construct was based on the findings that substitution of the BMP4 propeptide with that of BMP2 enhanced the secretion of mature BMP4 by the transduced cells. The ability of the BMP2/4 hybrid vector to enhance secretion of mature BMP4 was not cell-type-restrictive, as this BMP2/4 hybrid vector system enhanced secretion of mature BMP4 in normal rat marrow stromal cells and normal rat fibroblasts, as well as in transformed human 293T and HT1080 cells. The reason why replacement of the BMP4 propeptide with that of BMP2 improves the efficiency of mature BMP4 secretion in transduced cells is unclear. However, we should note that replacement of the propeptide domain of BMP2B with that of BMP2A also enhanced the expression and/or secretion of mature BMP2B in a plasmid vector-based system [25]. Moreover, the expression and secretion of VG1, another member of the TGF␤-BMP multigene family, in transduced cells was undetectable, unless a BMP propeptide domain was fused to the VG1 expression construct [30,31]. The fact that swapping the propeptide domain of several members of the TGF␤-BMP multigene family with that of other family members seemed to cause an enhancement in their expression and secretion in transduced cells raises the possibility that this feature might be unique to members

Two problems that limited the usefulness of retroviral vector systems to deliver a BMP gene, particularly BMP4, in the past have been the low level of secretion of BMP protein in transduced cells and the difficulty in generating stable BMP-expressing retroviral producer clones that would produce high-titer viruses. We have developed an MFG-based retroviral vector system that seems to overcome these limitations and, as such, could improve the usefulness of the retroviral vector-based system to deliver BMP4 for gene therapy of bone repair and regeneration. Accordingly, we showed that normal rat marrow stromal cells and transformed cell lines transduced with our retroviral vector produced and secreted levels of biologically active BMP4 protein in amounts often exceeding 1 ␮g/106 cells/24 hours. To our knowledge, this represents the first report of such a high level of secretion of biologically FIG. 7. Synthesis and secretion of BMP4 from normal rat dermal fibroblasts (RFS), noractive BMP4 with cells transduced with a retroviral mal RMSC, and HT1080 cells transduced with BMP2/4 retrovirus generated from three vector system. In addition, we were able to generate different HA-LB producer cell clones [1–3]. CM and cell lysate were analyzed two weeks post-transduction by SDS-PAGE and western blot. stable retroviral producer clones that yielded rela-

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FIG. 8. Ectopic bone formation in Fisher 344 rats, 4 weeks (A–C) or 12 weeks (D, E) after implantation with the pCLSABMP2/4transduced (A–D) or pCLSA␤-galtransduced (E) normal marrow stromal cells. (A–C) Evidence that bone nodules were present in sections from the implant site four weeks after transplantation. (A) Von Kossa staining demonstrates the presence of bone minerals in the nodules. (B) Toluidine blue staining of bone nodules demonstrates the presence of plump osteoblasts on the surface of the nodule. (C) ALP staining of bone nodule sections shows the presence of this marker enzyme on the surface of the osteoblasts and preosteoblasts surrounding the bone nodule, counter-stained with methyl green. (D) Toluidine blue staining of bone nodules in sections from the implant site 12 weeks after transplantation. Note that bone–marrow-like structures were present inside the bone nodule. (E) Toluidine blue staining of bone section from the implant site of control rats receiving the pCLSA␤-gal-transduced marrow stromal cells for 12 weeks.

of the TGF␤-BMP family. The molecular mechanism whereby the replacement of the propeptide of BMP4 by that of BMP2 caused an enhancement in secretion of mature BMP4 protein in transduced cells is not known. Because propeptide of a secretory protein often acts to enhance the stability of the protein, one may speculate that the propeptide of BMP2 could have a greater stabilization effect on BMP4 than the native BMP4 propeptide. If so, it follows that the increased amount of secreted mature BMP4 in CM of transduced cells could be due to an increased stability of the BMP2/4 fusion protein. Consistent with this speculation, it has been reported that the propeptide domain of dorsalin and nodal, two members of the TGF␤-BMP multigene family, has an important function in the stabilization of mature protein [32]. In this regard, the propeptide domain of dorsalin increased the stability of mature nodal, whereas the propeptide domain of nodal tended to destabilize nodal. That the swapping of the propeptide domain of BMP4 or dorsalin with that of nodal led to destabilization of mature BMP4 or dorsalin, respectively [32], lends further supports for the speculation. The third noteworthy characteristic of our MFG-based BMP4 retroviral vector system was the use of the amphotropic HA-LB packaging cells to produce stable cell clones producing retroviruses that express BMP2/4. There

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is evidence that the HA-LB packaging cells could have the following advantages to produce stable virus producer clones over the commonly used packaging cell lines. In our approach, we generated a high-titer VSV-G pseudotyped vector stock by a one-step, three-plasmid cotransfection into 293T cells. Using this high-titer, VSV-G pseudotyped stock, we not only eliminated the step of ecotropic packaging cell line establishment, but also eliminated the need for the marker gene selection in viral producer cell line. The design of retroviral constructs without the marker gene may prevent this unwanted complication for future in vivo experiments. Use of the HA-LB packaging cell line allowed us to generate several clones producing sufficiently high titers of BMP4-expressing retrovirus, indicating that HA-LB is an appropriate packaging cell line for producing stable BMPexpressing retrovirus producer clones. However, a previous study with a retroviral vector expressing BMP7 suggested that high titer of virus was toxic to the producer cells [20], a feature which might contribute to the inability to generate high-titer BMP-expressing retrovirus producer stable clones in the past. Our findings do not support this conclusion. There are several potential explanations for the apparent discrepancy. It is possible that BMP4 is less cytotoxic than BMP7 and, thus, high-titer BMP4 (but not BMP7) expressing retroviral producer clones can be generated.

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Conversely, it is also possible that the HA-LB cells are less sensitive to the cytotoxic effects of BMPs. Our study also disclosed an interesting property of the BMP4 protein. We found that the addition of a HA tag to the C terminus of human BMP4 protein completely abolished its biological activity. This was unexpected, because a previous study showed that a plasmid vector expressing mouse BMP4 with the same HA tag fused to the C terminus was able to enhance the healing of a segmental defect of femur in rat [33]. Although the biological activity of the mouse BMP4–HA fusion protein was not directly measured, their findings suggest that the mouse BMP4–HA fusion protein, unlike the human BMP4–HA fusion protein, was probably biologically active. We should emphasize that our results that human BMP4–HA protein lacks biological activity were extremely reproducible. The potential cause for the apparent discrepancy is unclear, but it may be related to different structural requirements between mouse and human BMP4 in that the C-terminal structure of human BMP4 may have a more essential role in its biological activity than that of mouse BMP4. We should also note that the BMP4–HA, although it is biologically inactive, did not seem to affect the biological activity of the native BMP4 in a dominant-negative manner, as co-expression of BMP4–HA and BMP4 expression vectors did not adversely affect the biological activity of the BMP4 (data not shown). Contrary to the C terminus, X-ray crystallography studies revealed that the N-terminal domain of human BMPs is relatively flexible [34], suggesting that the N terminus of human BMPs might have a lesser role in their biological activity. Consistent with this speculation is the finding that a Myc tag fused at the N-terminal end of human BMP4 had no significant deleterious effects on its biological activity [35]. Together, these findings suggest that a reporter tag may be added to the N terminus, but not the C terminus, of human BMP4 to aid in its identification and quantification. We have developed an MFG-based retroviral vector system that allows us to generate a high-titer BMP4 expressing retrovirus that could be used to transduce normal, as well as transformed, cells to express and secrete high levels of mature BMP4 protein. Most importantly, we demonstrated that normal rat marrow stromal cells transduced with our BMP4-expressing retrovirus induced ectopic bone formation in syngeneic immune competent rats. This in vivo finding, as well as our preliminary finding that this BMP4 retroviral system could successfully induce complete healing of calvarial critical size defects in the rat [36], indicate that this BMP4 retroviral vector system has therapeutic potential in gene therapy for bone repair and regeneration.

MATERIALS

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METHODS

Construction of BMP4 constructs and retroviral vectors. We amplified

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BMP4 and BMP2 cDNAs by PCR from the subcloned pGEM-BMP4 and pGEM-BMP2 human phage clones, respectively (ATCC, Manassas, VA). We introduced a Kozak sequence to the 5⬘ end of each cDNA to enhance protein expression in mammalian cells. We used BMP4N-1 (5⬘-CCGCTCGAGGCGGCCGCCCACCATGCTGATGGTCGTTTTATTATG-3⬘) and BMP4-2 (5⬘TCCATCGATAGATCTATCCTCAAGGACTGCCTG-3⬘) oligo primers to generate BMP4 expression construct. We used BMP4N-1 and BMP4N-2 (5⬘CGCGGATCCGTAGCGGCACCCACATCCCTCTAC-3⬘) primers to generate BMP4 tagged with a HA epitope (BMP4–HA). Similarly, we used BMP2N-1 (5⬘-CCGCTCGAGGCGGCCGCCCACCATGGTGGCCGGGACCCGCTGTCT3⬘) and BMP2-2 (5⬘-TCCATCGATAGATCTGCTGTACTAGCGACACCCACA3⬘) primers to amplify the BMP2 construct. Integrated DNA Technologies (Coralville, IA) synthesized all primers. To generate the BMP4–HA construct, we inserted a 27-bp double-stranded oligo (5⬘-GATCCTACCCATACGATGTTCCGGATTACGCTAGCCTCTAAAGATCTAT-3⬘; encoding the HA epitope) immediately upstream to the stop codon at the 3⬘ end of the BMP4 cDNA [24]. The Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) catalyzed all PCR reactions, using high template DNA concentrations (1 ␮g/100 ␮l) and low amplification cycles (20 cycles) to minimize PCR-introduced mutations. We introduced restriction sites (that is, a NotI site at the 5⬘ end and a BglII site at the 3⬘ end) to facilitate subcloning. We cloned all PCR products into the pBluescript II KS(–) phagemid. We verified sequence of selected clones by sequencing both strands of cDNA. We generated the BMP2/4 (or BMP2/4–HA) hybrid construct, in which the original sequence coding for the propeptide of BMP4 was replaced by the corresponding propeptide sequence of the BMP2 cDNA, as described [25]. Briefly, we replaced the 5⬘ end segment between the NotI site and the internal MscI site of the BMP4 cDNA with the corresponding NotI-MscI segment from the BMP2 cDNA with standard subcloning procedures. We generated retroviral vectors containing different BMP4 expression constructs by subcloning respective BMP4 constructs from the pBluescript II KS(-) into pLXSN, pLNCX, and pLNSX (obtained from A. Dusty Miller of Fred Hutchinson Cancer Research Center, Seattle, WA). We termed the resultant vectors pLBMP4SN, pLNCBMP4, and pLNSBMP4, respectively. For the MFG-based retroviral vector, we subcloned BMP2/4 and ␤-gal genes into pCLSA to generate pCLSABMP2/4 and pCLSA␤-gal vector plasmids, respectively. The backbone of this vector is essentially the same as that of pCLMFG-LacZ, but with one change [37]. The splicing acceptor site of our vector was derived from a 365-bp NdeI/XbaI fragment of the MLV-GP gene, and this fragment did not contain the AUG start codon of the MLV-env gene. The transcription of BMP2/4 and ␤-gal genes used the original AUG start codon of each respective gene. Cell isolation, culture, and transfection. We isolated bone marrow stromal cells from 5-week-old Fisher 344 male rats. We collected bone marrow cells by flushing the diaphyses and marrow cavity with sterile DMEM medium and cultured the resulting cells for 2 d in DMEM supplemented with 10% fetal bovine serum, streptomycin (100 ␮g/ml), and penicillin (100 U/ml). We washed the adhered cells three times with phosphate buffered saline (PBS) and grew the cells to confluence. We stored the cells in liquid nitrogen until use. The Animal Use Subcommittee of the Jerry L. Pettis Memorial VA Medical Center approved the animal protocol. We used the transient transfection approach to assess the ability of various BMP4 constructs to express mature BMP4 protein. We subcloned each BMP4 construct into pLNCX driven by the CMV promoter. We then transfected each vector into 293T cells by calcium phosphate precipitation [37]. We collected the conditioned medium (CM) and cell lysates of transfected cells, 24 h after the transfection, for BMP4 analysis. Retrovirus production and transduction. We used two methods to generate replication-defective retrovirus containing the BMP4 expression construct. The first method was based on transient transfection with three plasmids [38]: one encodes viral structural and enzymic proteins, the second encodes VSV-G (stomatitis vescular virus G protein), and the third encodes the transfer vector containing the BMP4 construct, pCLSABMP2/4 (or the reporter gene, pCLSA␤-gal). The three plasmids were cotransfected into 293T cells by calcium-phosphate precipitation [39]. We collected the CM containing viral particles 24 h post-transfection, filtered through a 0.45-␮m filter, and stored at –80oC until use. We determined viral titer of pCLSA␤-gal by end-point dilution as described [38]. Titers ranged from 1

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⫻ 106 to 5 ⫻ 106 transduction units per ml. For pCLSABMP2/4, as it has a backbone identical to that of pCLSA␤-gal, we estimated titer from the titer of pCLSA␤-gal, which generated in the same transient transfection experiments. The second method relied on the generation of a stable producer cell line using the amphotropic packaging cell line, HA-LB [29]. We seeded HA-LB cells (a gift of Chiron Technologies, San Diego, CA) at 2 ⫻ 105 cells/well in 6-well plates and infected the cells with 1.5 ml of various dilutions of the VSV-G pseudotyped viral stock. We added polybrene to a final concentration of 8 ␮g/ml. Forty-eight h after the transduction, we seeded the cells in 96-well plates at a low density of 1 cell/well, and screened selected cell clones for their ability to secrete high levels of BMP4 in HT1080 cells. We confirmed the ability of these clones to secrete high levels of BMP4 in normal rat marrow stromal cells. We collected viral stocks generated from the optimal producer clones, centrifuged the viral stocks at 2,000 rpm for 10 min at 4oC, and stored them at –80oC until use.

In vivo ectopic bone formation in rats. We anesthetized Fisher 344 male rats (5-week-old), after acclimation for a week, with Ketamine (50 ␮g/gm body weight) and Xylazine (5 ␮g/gm body weight). We made a 10-mm incision over the right lateral thoracic cage and created a subcutaneous pocket by blunt dissection under aseptic conditions. We implanted a gelfoam disk (Pharmacia & Upjohn, Peapack, NJ), impregnated with 2 ⫻ 106 transduced bone marrow cells and cultured overnight, into the subcutaneous pocket. We then sutured the wound and treated the wound with antibiotic ointments. We kept animals warm until they recovered from the anesthesia. We followed ectopic formation at the pocket every two weeks by radiographic examination using the Faxitron cabinet X-ray system (Faxitron, Wheeling, IL), and confirmed the ectopic formation with histology after the animals were euthanized at 4, 8, or 12 weeks post-implantation. The animal Use Committee of the Jerry L. Pettis Memorial VA Medical Center reviewed and approved the animal use procedure.

BMP4 biological activity assay. We used mouse C2C12 myoblastic cells in a bioassay to determine the biological activity of BMP4, because many laboratories also used these cells in similar assays to determine the biological activity of BMPs [40–43], and the supplier of our rhBMP4 (Research Diagnostics, Inc., Flander, NJ) also used a similar bioassay to determine the activity of BMP4 and showed that these cells responded to BMP4 in a dosedependent manner to increase alkaline phosphatase (ALP) expression. In this assay, we added an aliquot (100 ␮l) of serial twofold diluted samples (CM or cell lysates) in triplicate to the C2C12 cells, seeded at a density of 8 ⫻ 103 cells/well in 96-well plates. We included serially diluted rhBMP4 in each assay to construct the standard curve for comparison. After incubation for 24 h, we collected the cells for ALP staining, cell counting, and ALP enzymatic activity in 0.05% Triton X-100 cell extracts. We performed cell staining for ALP with the Sigma Diagnostics ALP kit (Sigma Diagnostics, Inc., St Louis, MO). We assayed ALP enzymatic activity for the production of p-nitrophenolate from p-nitrophenol phosphate [44] and reported the ALP activity as U/106 cells, where we defined 1 U as 1 ␮mole of product formed/min at room temperature. The increase in ALP activity in C2C12 cells in our assay was linear with the amount of rhBMP4 added in the assay (Fig. 3D).

Histological analysis. We harvested tissue samples at the time of euthanisia and fixed them in 10% neutral buffered formalin for 24 h. We divided each sample into two parts. We embedded one part undecalcified in glycol methacrylate. We demineralized and embedded the other part in paraffin. For glycol methacrylate embedding, we treated the samples successively with 70%, 85%, and 95% glycol methacrylate (JB4 kit, solution A, Polysciences, Inc., Warrington, PA) in the cold with shaking. We then infiltrated samples with glycol methacrylate containing 0.09% benzoyl peroxide for 2 d in the cold with shaking. We embedded samples in glycol methacrylate containing 2% solution B (JB4 kit) and 0.09% benzoyl peroxide in aluminum block holders and allowed the samples to polymerize under nitrogen in the cold. For paraffin embedding, we demineralized samples in 0.1% sodium citrate in 22.5% formic acid and dehydrated samples in graded levels of alcohol and embedded them in paraffin. We stained sections cut from the glycol methacrylate blocks with Goldner’s Trichrome stain or by Von Kossa. We stained sections cut from paraffin blocks with Toluidine blue or hematoxylin and eosin, or for ALP staining.

Western blot analysis. We denatured CMs from transfected or transduced cells at 100⬚C for 5 min in 3 volumes of SDS PAGE-sample buffer containing 4% SDS, 10% ␤-mercaptoethanol and 10 mM Tris (pH 8.0). We fractionated the denatured samples through a 12% polyacrylamide-SDS gel and transblotted them onto a 0.2-␮m PVDF membrane (Biorad, Hercules, CA). We blocked the membrane with 1% skim milk for 90 min, incubated the membrane for 90 min with 1 ␮g/ml monoclonal anti-human BMP4 antibody (R & D, St. Paul, MN). After washing, we incubated the membrane for 90 min with 1:1000-diluted HRP-labeled goat anti-mouse IgG (Pierce, Rockford, IL), incubated it with 1:10 diluted SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 3–5 min, and exposed it to a Kodak X-Omat film. We estimated the level of BMP4 protein from the relative density of the BMP-4 band (determined by laser densitometry) compared with that of a known amount of rhBMP4 on the same blot. We concentrated CM containing BMP4 that was below the sensitivity of detection (that is, 50 ng/ml) 10-fold with Centricon YM-10 before re-assay. To measure the cellular levels of BMP4, we lysed cells with 200 ␮l/well of the SDS-PAGE sample buffer, and we again measured BMP4 by western blot. We counted the cells in replicate wells to determine the number of cells. In situ immunocytochemical staining of BMP4. We washed cells twice with cold PBS, fixed them in cold acetone/methanol (1:1 v/v) for 5 min at –20oC, and air-dried them. We treated the fixed cells for 30 min at room temperature with 0.6% H2O2 in PBS to quench endogenous peroxidase activity. After washing the cells twice with PBS, we blocked the cells for 2 h at room temperature with 5% normal goat serum and incubated them at 4oC overnight with the 1:100-diluted goat anti-BMP4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). We added the biotinylated secondary antibody to the cells, rinsed them with PBS, and added to them HRPstreptavidin complex. After rinsing the cells once with PBS, we incubated the cells with the HRP substrate as recommended by the supplier (Goat ImmunoCruz Staining System, Santa Cruz Biotechnology).

MOLECULAR THERAPY Vol. 4, No. 2, August 2001 Copyright © The American Society of Gene Therapy

ACKNOWLEDGMENTS We thank M. Bhattacharyya, S. Baazizi, J. Halter, N. Lowen, and Y.-H. Liu for technical assistance; X. S. Zhang, R. Gysin, and J. Smallwood for help with animal experiments; and S. Mohan and D. Strong for suggestions and discussion. This material is based on work supported by a special appropriation to the Jerry L. Pettis Memorial VA Medical Center–Musculoskeletal Disease Center. All work was performed with facilities provided by the Department of Veterans Affairs. RECEIVED FOR PUBLICATION MARCH 14; ACCEPTED JUNE 13, 2001.

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